Mechanical stimuli play a major role in the regulation of skeletal muscle mass, and the maintenance of muscle mass contributes significantly to disease prevention and quality of life. Although the link between mechanical signals and the regulation of muscle mass has been recognized for decades, the molecular mechanisms that drive this process are still not known. Hence, the long-term goal of our research is to define the molecular events via which mechanical stimuli regulate skeletal muscle mass. The primary objective of this project is to determine the extent to which changes in the phosphorylation of TRIM28 contribute to the mechanical regulation of muscle mass. We are focusing on this topic because TRIM28 can control the activity of mTOR (a kinase that has been widely implicated in the mechanical regulation of muscle mass). A recent study also identified TRIM28 as a scaffold protein that interacts with key myogenic transcription factors (e.g., Mef2 and MyoD), and it has been shown that phosphorylation of the S473 residue on TRIM28 can act as a switch that unleashes the transcriptional activity of Mef2 and MyoD. This is intriguing because alterations in the activity of MyoD and Mef2 have been widely implicated in the regulation of muscle mass, and a recent phosphoproteomic analysis from our lab revealed that mechanical stimulation leads to a profound increase in TRIM28(S473) phosphorylation. Moreover, we discovered that the expression of a S473 phosphomimetic mutant of TRIM28 is sufficient to induce hypertrophy, and that the hypertrophic effect is dependent on the phosphomimetic mutation. Combined, these observations led us to our central hypothesis: an increase in TRIM28(S473) phosphorylation is a fundamental part of the pathway via which mechanical stimuli promote an increase in muscle mass. To rigorously test this hypothesis, we will first use of a combination of biochemical, molecular and genetic interventions in mice. Importantly, the mouse-based studies will enable us to: i) gain insight into the mechanisms via which TRIM28(S473D) induces hypertrophy, and ii) define the role that both myofiber and satellite cell specific changes in TRIM28(S473) phosphorylation play in mechanical load-induced hypertrophy. In addition to the mouse-based studies, we will also perform a human trial to determine whether the primary conclusions from mice can be translated to the human condition. Collectively, the outcomes of this project are expected to establish TRIM28 as a novel regulator of muscle mass and shed light on some of the basic mechanisms through which alterations in S473 phosphorylation control its hypertrophic effect. The outcomes are also expected to reveal the existence of a TRIM28-dependent pathway that not only enables mechanical stimuli to induce hypertrophy, but also the activation of satellite cell proliferation and fusion. Such outcomes would not only dramatically advance our understanding of how mechanical stimuli regulate muscle mass, but they would also create a new landmark for future studies that are aimed at developing a comprehensive understanding of this highly important process.